The Cystic Fibrosis Transmembrane Conductance Regulator Activates Aquaporin 3 in Airway Epithelial Cells

Enhanced osmotic water permeability has been observed in Xenopus oocytes expressing cystic fibrosis transmembrane conductance regulator (CFTR) protein. Subsequent studies have shown that CFTR activates an endogenous water permeability in oocytes, but that CFTR itself is not the water channel. Here, we show CFTR-dependent activation of endogenous water permeability in normal but not in cystic fibrosis human airway epithelial cells. Cell volume was measured by novel confocal x-z laser scanning microscopy. Glycerol uptake and antisense studies suggest CFTR-dependent regulation of aquaporin 3 (AQP3) water channels in airway epithelial cells. Regulatory interaction was confirmed by coexpression of CFTR and AQP3 cloned from human airways inXenopus oocytes and of CFTR and rat AQP3 in Chinese hamster ovary cells. These findings indicate that CFTR is a regulator of AQP3 in airway epithelial cells. Enhanced osmotic water permeability has been observed in Xenopus oocytes expressing cystic fibrosis transmembrane conductance regulator (CFTR) protein. Subsequent studies have shown that CFTR activates an endogenous water permeability in oocytes, but that CFTR itself is not the water channel. Here, we show CFTR-dependent activation of endogenous water permeability in normal but not in cystic fibrosis human airway epithelial cells. Cell volume was measured by novel confocal x-z laser scanning microscopy. Glycerol uptake and antisense studies suggest CFTR-dependent regulation of aquaporin 3 (AQP3) water channels in airway epithelial cells. Regulatory interaction was confirmed by coexpression of CFTR and AQP3 cloned from human airways inXenopus oocytes and of CFTR and rat AQP3 in Chinese hamster ovary cells. These findings indicate that CFTR is a regulator of AQP3 in airway epithelial cells. cystic fibrosis transmembrane conductance regulator wild-type CFTR cystic fibrosis Chinese hamster ovary aquaporin human AQP rat AQP reverse transcription-polymerase chain reaction isobutylmethylxanthine The cystic fibrosis transmembrane conductance regulator (CFTR)1 protein is mutated and defective in cystic fibrosis (CF), a common lethal genetic disease. CFTR has been demonstrated to function in two ways: (i) as a protein kinase A-regulated Cl− channel and (ii) as a regulator of other membrane conductances (1Stutts M.J. Canessa C.M. Olsen J.C. Hamrick M. Cohn J.A. Rossier B.C. Boucher R.C. Science. 1995; 269: 847-850Crossref PubMed Scopus (958) Google Scholar, 2Riordan J.R. Annu. Rev. Physiol. 1993; 55: 609-630Crossref PubMed Scopus (307) Google Scholar, 3Greger R. Mall M. Bleich M. Ecke D. Warth R. Riedemann N. Kunzelmann K. J. Mol. Med. 1996; 74: 527-534Crossref PubMed Scopus (48) Google Scholar). Thus, CF may not only affect cAMP-dependent Cl− conductance, but may also affect other membrane conductances normally regulated by wild-type CFTR. Such a defect in CFTR-dependent regulation was found for the epithelial Na+ conductance (1Stutts M.J. Canessa C.M. Olsen J.C. Hamrick M. Cohn J.A. Rossier B.C. Boucher R.C. Science. 1995; 269: 847-850Crossref PubMed Scopus (958) Google Scholar, 4Mall M. Hipper A. Greger R. Kunzelmann K. FEBS Lett. 1996; 381: 47-52Crossref PubMed Scopus (132) Google Scholar). Thus, enhanced Na+ conductance was detected in the airways and intestinal epithelium of CF patients, which is very likely to contribute to enhanced absorption of electrolytes and water and to the altered mucociliary clearance as well as intestinal obstructions, respectively (5Littlewood J.M. Br. Med. Bull. 1992; 48: 847-859Crossref PubMed Scopus (56) Google Scholar, 6Boucher R.C. Cotton C.U. Gatzy J.T. Knowles M.R. Yankaskas J.R. J. Physiol. (Lond.). 1988; 405: 77-103Crossref Scopus (202) Google Scholar, 7Mall M. Bleich M. Greger R. Schreiber R. Kunzelmann K. J. Clin. Invest. 1998; 102: 15-21Crossref PubMed Scopus (185) Google Scholar). Apart from epithelial Na+ conductance, osmotic water permeability has been reported to be influenced by CFTR (8Hasegawa H. Skach W. Baker O. Calayag M.C. Lingappa V. Verkman A.S. Science. 1992; 258: 1477-1479Crossref PubMed Scopus (125) Google Scholar, 9Schreiber R. Greger R. Kunzelmann K. Pfluegers Arch. Eur. J. Physiol. 1997; 434: 841-847Crossref PubMed Scopus (44) Google Scholar). These studies were performed in Xenopus oocytes and indicated enhanced osmotic cell swelling after expression and cAMP-dependent activation of CFTR. Since water-injected control oocytes did not demonstrate such a cAMP-activated water permeability, the most likely explanation was that cAMP acted through activation of CFTR. In fact, it was suggested initially that water uses the same conductive pathway and moves together with Cl−through the CFTR Cl− channel. This pathway should therefore be formed by transmembrane domains (8Hasegawa H. Skach W. Baker O. Calayag M.C. Lingappa V. Verkman A.S. Science. 1992; 258: 1477-1479Crossref PubMed Scopus (125) Google Scholar). However, it was shown in a subsequent study (a) that both water and Cl− ions moved independently when CFTR was activated by an increase in intracellular cAMP. Independence was demonstrated by showing selective inhibition of CFTR Cl− conductance by glibenclamide and selective blockage of water permeability by mercurial compounds and phloretin (9Schreiber R. Greger R. Kunzelmann K. Pfluegers Arch. Eur. J. Physiol. 1997; 434: 841-847Crossref PubMed Scopus (44) Google Scholar). In addition, this water pathway activated by CFTR was permeable for glycerol, too. These results support the assumption that some sort of endogenous water and glycerol permeability must be present in Xenopus oocytes and that this is activated by CFTR. So far, it was unclear whether CFTR-activated water permeability is unique to Xenopus oocytes or whether a similar interaction can also be observed in mammalian cells. This question was addressed in this study by measuring cell volume changes and radioactive glycerol uptake in human airway epithelial and Chinese hamster ovary (CHO) cells. We found CFTR-dependent activation of osmotic water permeability in normal respiratory cells and identified one member of the aquaporin family (AQP3) as the interacting partner. Since CFTR-dependent stimulation of osmotic water permeability is absent in airway cells derived from CF patients, we speculate that this has a pathophysiological impact on the lung disease in CF. Human non-CF, CF, and CF airway epithelial cells transfected with 6REP-wtCFTR have been described in previous studies (10Cozens A.L. Yezzi M.J. Kunzelmann K. Ohrui T. Chin L. Eng K. Finkbeiner W.E. Widdicombe J.H. Gruenert D.C. Am. J. Respir. Cell Mol. Biol. 1994; 10: 38-47Crossref PubMed Scopus (777) Google Scholar, 11Lei D.C. Kunzelmann K. Koslowsky T. Yezzi M.J. Escobar L.C. Xu Z.D. Rommens J.M. Tsui L.-C. Tykocinski M. Gruenert D.C. Gene Ther. 1996; 3: 427-436PubMed Google Scholar) and were kindly provided by Dr. D. C. Gruenert (University of California, San Francisco). CHO-K1 cells and CHO cells stably expressing wtCFTR (CHO-wtCFTR) or ΔF508-CFTR (CHO-ΔF508) were kindly provided by Dr. X.-B. Chang (Mayo Clinic, Scottsdale, AZ). Cells were cultured on tissue culture plastic dishes or glass coverslips and kept in an atmosphere of 5% CO2 under conditions described elsewhere (10Cozens A.L. Yezzi M.J. Kunzelmann K. Ohrui T. Chin L. Eng K. Finkbeiner W.E. Widdicombe J.H. Gruenert D.C. Am. J. Respir. Cell Mol. Biol. 1994; 10: 38-47Crossref PubMed Scopus (777) Google Scholar, 12Tabcharani J.A. Chang X.-B. Riordan J.R. Hanrahan J.W. Nature. 1991; 352: 628-631Crossref PubMed Scopus (455) Google Scholar). CHO cells were transfected with rat AQP3 cDNA (kindly provided by M. Echevarrı́a, University of Sevilla, Sevilla, Spain) inserted into expression vector pZeoSV (Invitrogen) using standard techniques. Isolated colonies were expanded and assayed for AQP3 expression by measurement of glycerol uptake and detection of AQP3 mRNA (RT-PCR). Cells grown on coverslips were loaded with the fluorescent dye calcein/AM (10 μmol/liter for 30 min at room temperature), and repetitive x-z line scans at 488 nm excitation of one scanning line through three to six cells were performed using an LSM 410 apparatus (Zeiss, Germany) (13Nitschke R. Benning N. Ricken S. Leipziger J. Fischer K. Greger R. Pfluegers Arch. Eur. J. Physiol. 1997; 434: 466-474Crossref PubMed Scopus (14) Google Scholar). A water immersion lens (Zeiss C-APO 63/1.2w) was used to avoid optical distortions due to refractive index mismatches. The z-line scan distance was set to result in square voxel; usually these were in z-steps of 0.33 μm. The size of the confocal pinhole was set to give a full-width half-maximum in the z-direction of 0.4 μm. The z-line scans were stacked in a two-dimensional image giving a x-z slide through the cells. A customized macro allowed the recording and storage of time series of x-z slides (usually 200–300 images). Cell area and the mean intensity of the cell area at the cutting z-line were analyzed with Metafluor software (Universal Imaging Corp., West Chester, PA). Initial area increase and initial intensity decrease induced by hypotonic bath solution due to omission of mannitol (72.5 mmol/liter NaCl, 0.4 mmol/liter KH2PO4, 1.6 mmol/liter K2HPO4, 1 mmol/liter MgCl2, 1.3 mmol/liter calcium gluconate, and 5 mmol/liter d-glucose, pH 7.4) were determined in the same cells before and after stimulation and in the presence of IBMX (0.5 mmol/liter) and forskolin (10 μmol/liter). The number of cells was assessed, and cells were incubated in a 14Cglycerol-containing solution consisting of 160 mmol/liter 14Cglycerol (final activity = 1 mCi/liter or 37 MBq/liter), 65 mmol/liter NaCl, 1.6 mmol/liter K2HPO4, 0.4 mmol/liter KH2PO4, 1.3 mmol/liter CaCl2, 1 mmol/liter MgCl2, and 5 mmol/liter d-glucose, pH 7.4, at 37 °C for 1, 2, and 3 min. Cells were rinsed three times with ice-cold unlabeled glycerol solution and lysed in 100 g/liter SDS at room temperature. Radioactivity was measured by liquid scintillation counting. Glycerol uptake was measured in the presence of IBMX (0.5 mmol/liter) and forskolin (10 μmol/liter) and in cells preincubated with 5 μmol/liter HgCl2 for 15 min. Total RNA of non-CF and CF cells was used for RT-PCR analysis of AQP1, AQP3, AQP4, and AQP5 expression. The following primers were used: AQP1, 5′-ATGGCCAGCGAGTTCAAGA-3′ (sense) and 5′-TAGTAGCCAGCACGCATAG-3′ (antisense); AQP3, 5′-ATGGGTCGACAGAAGGAG-3′ (sense) and 5′-CTCAGATCTGCTCCTTGT-3′ (antisense); AQP4, 5′-ATGGATGCTGAGGTGCCA-3′ (sense) and 5′-CACACACTCTCCATCTCC-3′ (antisense); and AQP5, 5′-ATGAAGAAGGAGGTGTGC-3′ (sense) and 5′-TCATCTCCAGGGAGCCAG-3′ (antisense). The 879-base pair coding sequence of human AQP3 was RT-PCR-amplified from non-CF cells using 5′-CCGCCATGGGTCGACA-3′ (sense) and 5′-CTCAGATCTGCTCCTTGT-3′ (antisense). Resulting PCR products were sequenced (Applied Biosystems Model 373A). For expression inXenopus oocytes, cDNA encoding human AQP3 was subcloned into oocyte expression vector pTLN, which uses the Xenopusβ-globin untranslated regions to boost expression in oocytes (14Lorenz C. Pusch M. Jentsch T.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13362-13366Crossref PubMed Scopus (215) Google Scholar). Airway cells and CHO cells grown on glass coverslips were fixed with methanol/acetone/Formalin solution (45:45:5) for 90 s. After washing with TBS-P (1 liter of 0.9 g of Tris base, 6.85 g of Tris-HCl, 8.78 g of NaCl, and 0.1% Tween 20, pH 7.5), cells were incubated with antibodies against AQP3 (1 μg/ml; kindly provided by M. A. Knepper, National Institutes of Health, Bethesda, MD) for 30 min at room temperature. All antibodies were diluted in RPMI 1640 medium (Sigma R 1145) with 10% heat-inactivated bovine serum and 0.1% sodium acid. After washing with TBS-P, cells were incubated with mouse anti-rabbit IgG (1:50; Dako M0737) for 30 min, washed with TBS-P, incubated with rabbit anti-mouse IgG (1:25; Dako Z0259) for 30 min, washed again, and incubated with APAAP complex (1:50; Dako D0651) for 30 min. Peroxidase reaction was visualized by naphthol AS-BI phosphate (Sigma N 2250) and new fuchsin (Merck). The reaction was stopped with double-distilled H2O, and cells were counterstained with Mayer's hematoxylin solution and embedded in Kaiser's glycerol gelatin. Subconfluent (70%) non-CF and CF-wtCFTR cells were incubated with 20 μmol/liter phosphorothioated sense and antisense oligonucleotides (AQP3, 5′-CAGCTCCTTCTGTCGACCCAT-3′ (antisense) and 5′-ATGGGTCGACAGAAGGAGCTG-3′ (sense); AQP1, 5′-CTTCTTGAACTCGCTGGCCAT-3′ (antisense); and AQP5, 5′-GGAGCACACCTCCTTCTT-CAT-3′ (antisense)) in serum-free culture medium supplemented with 2% Ultroser G (Life Technologies, Inc.) for 48 h, and subsequently, glycerol uptake was measured. Isolation and microinjection of oocytes have been described in a previous report (15Hipper A. Mall M. Greger R. Kunzelmann K. FEBS Lett. 1995; 374: 312-316Crossref PubMed Scopus (45) Google Scholar). Oocytes of identical batches were injected with 10–50 ng of cRNA (wild-type CFTR, G551D-CFTR, and hAQP3 cloned from human airway cells). Water-injected oocytes served as controls. The osmotic water permeability coefficient was from the of hypotonic volume measured by at °C (9Schreiber R. Greger R. Kunzelmann K. Pfluegers Arch. Eur. J. Physiol. 1997; 434: 841-847Crossref PubMed Scopus (44) Google Scholar). was induced by omission of mmol/liter mannitol from was to M. S. J. J. Physiol. 1993; PubMed Scopus Google = the oocyte the and the at The was from the 1 min after to hypotonic Glycerol permeability was from the initial of 14Cglycerol uptake oocyte with 14Cglycerol and 1 mmol/liter Oocytes were lysed in 100 g/liter SDS at room and was All used were the of as to the number of and were used for with of permeability in airway epithelial cells was by measuring induced cell swelling using the novel described (13Nitschke R. Benning N. Ricken S. Leipziger J. Fischer K. Greger R. Pfluegers Arch. Eur. J. Physiol. 1997; 434: 466-474Crossref PubMed Scopus (14) Google Scholar). to hypotonic bath solution in an increase in cell area and a decrease in as shown for a non-CF cell in was to the the 30 after to the hypotonic bath solution 1 with IBMX and forskolin the area increase and the decrease by hypotonic bath These measured volume changes the of hypotonic cell swelling and the of CFTR on osmotic water permeability the cells volume decrease which be when CFTR Cl− conductance is activated (9Schreiber R. Greger R. Kunzelmann K. Pfluegers Arch. Eur. J. Physiol. 1997; 434: 841-847Crossref PubMed Scopus (44) Google Scholar). The shown in A that cell swelling was enhanced after activation of CFTR Such a cAMP-dependent increase in hypotonic cell swelling was not observed in airway epithelial cells derived from CF of CFTR water permeability in non-CF but not CF airway epithelial cells. shown is a of cell volume in non-CF cells. by IBMX and forskolin enhanced initial area increase and initial decrease in cells with control conditions before and after stimulation = CFTR-activated water permeability to an increase in the intensity was = in CF epithelial stimulation by IBMX and forskolin on cell volume = indicate from control studies have demonstrated that osmotic water permeability activated by CFTR in Xenopus oocytes is also permeable for we measured 14Cglycerol uptake in airway epithelial cells that endogenous wild-type CFTR CFTR (CF), or wild-type CFTR After stimulation of non-CF cells with IBMX and 14Cglycerol uptake was enhanced when with control uptake Such an activation of 14Cglycerol uptake by cAMP was not observed in CF cells. Here, 14Cglycerol uptake was when the cAMP-dependent pathway was However, expression of wild-type CFTR in CF cells the of IBMX and of the cells with HgCl2 15 the of cAMP on 14Cglycerol uptake in non-CF cells as well as in CF cells expressing CFTR this CFTR Cl− were not not These results in with has been observed in Xenopus oocytes (9Schreiber R. Greger R. Kunzelmann K. Pfluegers Arch. Eur. J. Physiol. 1997; 434: 841-847Crossref PubMed Scopus (44) Google also in human airway epithelial a water channel is activated by CFTR. permeability in is in cells by water Annu. Rev. Physiol. 1996; PubMed Scopus Google Scholar). of AQP3, AQP4, and in non-CF and CF airway epithelial cells was performed by expression of be detected in airway epithelial cells. However, expression of AQP1, AQP3, and AQP5 was found not for AQP3 was detected in airway epithelial cells from as well as in cultured CF and non-CF airway epithelial cells Since AQP3, in to or AQP5, has been described to glycerol K. S. K. S. M. H. T. K. T. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google M. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google these results suggest that AQP3 is the water pathway for CFTR-dependent water and glycerol This was by of AQP3 in these cells. of AQP3 demonstrated expression of this protein the membrane AQP3 was also in CHO cells expressing AQP3 and in CF airway but not in control CHO cells or in the of the normal respiratory epithelial cells expressing endogenous CFTR and CF epithelial cells expressing wild-type CFTR were incubated with AQP3 sense and antisense After 48 of glycerol uptake was in non-CF and CF-wtCFTR cells. such on glycerol uptake were observed in cells incubated with sense oligonucleotides for AQP3 or antisense oligonucleotides for and AQP5 that AQP3 is for CFTR-dependent activation of osmotic water permeability in airway epithelial cells. Human AQP3 was cloned from human airway epithelial cells used by The coding sequence was identical to the sequence in a previous report K. S. T. 1995; PubMed Scopus Google Scholar). of the cloned hAQP3 cDNA in Xenopus oocytes induced an enhanced osmotic glycerol permeability coefficient (9Schreiber R. Greger R. Kunzelmann K. Pfluegers Arch. Eur. J. Physiol. 1997; 434: 841-847Crossref PubMed Scopus (44) Google Scholar) = with control oocytes = CFTR and hAQP3 were oocytes, enhanced by stimulation of the oocytes with IBMX increase was observed when hAQP3 was not in oocytes, activation of CFTR by IBMX in the presence of an bath solution induced initial cell due to Cl− from the oocytes Subsequent to an hypotonic solution in the presence of IBMX cell swelling and the osmotic water permeability coefficient activation of hAQP3 water channels by CFTR A and described for the performed in airway the detected volume changes the impact of CFTR on osmotic water permeability of the volume decrease that is by activation of CFTR Cl− conductance (9Schreiber R. Greger R. Kunzelmann K. Pfluegers Arch. Eur. J. Physiol. 1997; 434: 841-847Crossref PubMed Scopus (44) Google Scholar). A in the of CFTR the of IBMX on regulation of hAQP3 by of CFTR. The results in Xenopus oocytes have been in a mammalian expression by and wtCFTR or in CHO cells. Glycerol uptake was measured before and after stimulation with forskolin and shown in control cells or cells expressing only wtCFTR or ΔF508-CFTR demonstrated 14Cglycerol which was not influenced by stimulation with forskolin and and CHO cells were transfected with and expression was detected by RT-PCR analysis not expression of enhanced glycerol uptake in three cells to and cells did not glycerol uptake to IBMX and it was in cells the 14Cglycerol uptake by expression of and the uptake by activation of wtCFTR were by HgCl2 μmol/liter). These indicate CFTR-dependent activation of hAQP3 when both in CHO cells. The present show for a novel of CFTR-dependent regulation of epithelial membrane demonstrate that CFTR, when by protein kinase activates a water permeability in respiratory epithelial cells. These results suggest Cl− as performed by the CFTR and water water permeability to in the airway In that this to of the as well as mucociliary clearance J.H. Widdicombe Respir. Physiol. 1995; PubMed Scopus Google Scholar, R.C. Knowles M.R. M.J. Gatzy J.T. Scopus Google Scholar). of in the AQP1, AQP3, AQP4, and AQP5 S. Am. J. Physiol. 1997; PubMed Google Scholar). AQP expression in the lung a complex of the which to be in the as well as in the lung S. Am. J. Physiol. 1997; PubMed Google Scholar). AQP3 is in of cells the S. Am. J. Physiol. 1997; PubMed Google Scholar). These as the for S. Am. J. Physiol. 1997; PubMed Google Scholar, Yankaskas J.R. Boucher R.C. Cohn J.A. J.M. 1992; PubMed Scopus Google Scholar, C. Finkbeiner W.E. Widdicombe J.H. J. Physiol. (Lond.). 1997; Scopus Google Scholar). the other AQP3 is in cells of the and in of epithelial cells S. Am. J. Physiol. 1997; PubMed Google Scholar, S. Am. J. Physiol. 1997; PubMed Google Scholar) in of electrolytes R.C. Cotton C.U. Gatzy J.T. Knowles M.R. Yankaskas J.R. J. Physiol. (Lond.). 1988; 405: 77-103Crossref Scopus (202) Google Scholar, J.H. Widdicombe Respir. Physiol. 1995; PubMed Scopus Google Scholar). In this expression of AQP3 and CFTR-dependent regulation were detected in cultured airway epithelial which demonstrate of cell such as expression of Na+ (10Cozens A.L. Yezzi M.J. Kunzelmann K. Ohrui T. Chin L. Eng K. Finkbeiner W.E. Widdicombe J.H. Gruenert D.C. Am. J. Respir. Cell Mol. Biol. 1994; 10: 38-47Crossref PubMed Scopus (777) Google K. S. Hipper A. Gruenert D.C. Greger R. Pfluegers Arch. Eur. J. Physiol. 1996; PubMed Scopus Google Scholar). However, expression of AQP3 not to be to the of these the of cells that of cells. the respiratory CFTR is in of cells to a also in of epithelial cells. CFTR Cl− can also be detected in cells K. S. Greger R. Pfluegers Arch. Eur. J. Physiol. 1995; PubMed Scopus Google but is for CFTR expression in of cell M. Cohn J.A. Yankaskas J.R. J.M. J. Clin. Invest. 1994; 93: PubMed Scopus Google Scholar). This for the CFTR and AQP3 on the of airway cells. We may therefore only speculate in cell of the respiratory the interaction studies have to whether AQP3 to both airway absorption and and it in of the airway which is for mucociliary CFTR-dependent activation of AQP3 not in airway cells the CF defect or in cells both AQP3 and of CFTR. We may therefore speculate that regulation of AQP3 by CFTR is in the respiratory of CF patients, which may contribute to the and absorption detected in CF airways R.C. Cotton C.U. Gatzy J.T. Knowles M.R. Yankaskas J.R. J. Physiol. (Lond.). 1988; 405: 77-103Crossref Scopus (202) Google Scholar, C. Finkbeiner W.E. Widdicombe J.H. J. Physiol. (Lond.). 1997; Scopus Google Scholar, Yankaskas J. J. Am. J. Physiol. 1996; PubMed Google Scholar). of AQP3 by CFTR is for CFTR-dependent regulation of other epithelial membrane other conductances have been reported to be regulated by CFTR, the epithelial Na+ conductance and conductance Cl− channel Cl− channel and conductance (1Stutts M.J. Canessa C.M. Olsen J.C. Hamrick M. Cohn J.A. Rossier B.C. Boucher R.C. Science. 1995; 269: 847-850Crossref PubMed Scopus (958) Google Scholar, 4Mall M. Hipper A. Greger R. Kunzelmann K. FEBS Lett. 1996; 381: 47-52Crossref PubMed Scopus (132) Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). the of channels and for the glibenclamide is enhanced by CFTR C.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). The of this regulation under R. Mall M. Bleich M. Ecke D. Warth R. Riedemann N. Kunzelmann K. J. Mol. Med. 1996; 74: 527-534Crossref PubMed Scopus (48) Google Scholar). in most for CFTR-dependent inhibition of the epithelial Na+ conductance, which was shown only to in the respiratory tissue M. Bleich M. Greger R. Schreiber R. Kunzelmann K. J. Clin. Invest. 1998; 102: 15-21Crossref PubMed Scopus (185) Google Scholar). CFTR-dependent regulation of epithelial on the presence of Cl− ions and the of an of CFTR. in membrane a interaction of both M. 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